A tight balance between bone resorption by osteoclasts and bone formation by osteoblasts is required for bone homeostasis. In a pathological context, enhanced osteoclastic bone resorption leads to a reduction of bone mass, as seen in osteoporosis, while defects in either osteoclast differentiation or function result in an abnormal accumulation of bone, as observed in osteopetrosis. Genetic studies of patients with osteopetrosis and naturally occurring or targeted gene knockout in rodent osteopetrotic models have unveiled important regulatory mechanisms of osteoclastic bone resorption. Disruption of genes such as c-src, [unreadable]3 integrin subunit, cathepsin K, TCIRG1 (encoding a3 subunit of vacuolar H+-ATPase), CLCN7 (encoding clc-7 chloride channel) and OSTM1 (grey lethal) disrupts osteoclast function. Although mutations in TCIRG1 and CLCN7 are most abundant in human osteopetrosis patients, the involved genes have not been identified in about 25% of cases. Recently, PLEKHM1 was identified as a gene mutated in the osteopetrotic ia/ia (incisors absent) rat and a subset of patients with osteopetrosis. Osteoclasts from ia/ia rats are dysfunctional due to impaired ruffled border formation and secretion. However, the detailed mechanisms by which PLEKHM1 regulates osteoclast function remain unknown. We find that 1) expression of PLEKHM1 increases during osteoclast differentiation 2) knockdown of PLEKHM1 by shRNA inhibits cathepsin K secretion and osteoclast function, without a decrease in osteoclast formation and 3) plekhm1 binds to TRAF6 and rab7 in mature osteoclasts, two proteins critical for the cell's capacity to resorb bone. Thus, we hypothesize that plekhm1 regulates osteoclast function, at least in part, through interaction with TRAF6 and rab7. Since we have developed a retroviral system by which we can knockdown an endogenous gene and simultaneously rescue it with an RNAi-resistant mutant, we are in a position to explore the role of plekhm1 in osteoclast function by performing structure/function analysis. Therefore, our specific aims are to identify the domain or motif in plekhm1 essential for 1) plekhm11/TRAF6 interaction and osteoclast function and 2) rab7 binding and osteoclast function. The osteoclast, a cell found exclusively in bone, is responsible for dissolution of this important tissue. The aging American population continues to lose bone because of osteoclast activity, leading to major public health problems that currently cost in excess of $15 billion dollars annually. We have identified a novel protein in osteoclasts which contributes to their bone resorbing activity. We plan to determine how this new protein regulates the osteoclast, raising the possibility that we will be able to develop a new drug that prevents bone loss.